Removal of Transgenes and Evaluation of Yield Penalties in Genome Edited Bacterial Blight Resistant Rice Varieties.

ABSTRACTBacterial blight (BB) of rice, caused byXanthomonas oryzaepv.oryzae(Xoo) is one of the major drivers of yield losses in Africa and Asia. Xoo secretes TAL-effectors (TALe) that induce host SWEET sucrose uniporter by binding to the effector binding element (EBE) ofSWEETpromoters, likely required for Xoo reproduction and virulence. We had multiplex edited the EBEs of three SWEET genes to prevent TALe binding, producing genome-edited (GE’d) rice mega-varieties (IR64, Ciherang-Sub1 for Asia, and Komboka for Africa) that were resistant to a wide spectrum of Xoo strains. Here, we report comprehensive analyses of the GE’d lines, including evaluation of agronomic performance in multi-location multi-season experimental field plots under different fertilization regimes, and tests for the presence/absence of foreign DNA/transgene in the offspring of GE’d lines (IR64-BC1T6, Ciherang-Sub1-BC1T5, Komboka-T3). Various strategies were evaluated, including herbicide tolerance, PCR, DNA gel blotting, whole genome sequencing (WGS), and specific tests stipulated by country-specific biosafety guidelines. Different WGS technologies were evaluated and also used to identify heritability of the edits, single nucleotide polymorphisms (SNPs), and insertions/deletions (indels) that might have resulted from somaclonal variation and potential GE-induced off-target mutations. Complete genome reference sequences for the parental lines IR64, Ciherang-Sub1, and Komboka are provided. In the field experiments, the GE’d lines did not show performance defects. Together, the results indicate that select GE lines do not contain foreign DNA or transgene fragments and fulfill the requirements for treatment equivalent to classical breeding lines in countries such as India and Kenya.

Basmati rice is famous around the world for its flavor, aroma, and long grain. Its demand is increasing worldwide, especially in Asia. However, its production is threatened by various problems faced in the fields, resulting in major crop losses. One of the major problems is bacterial blight caused by Xanthomonas oryzae pv. oryzae (Xoo). Xoo hijacks the host machinery by activating the susceptibility genes (OsSWEET family genes), using its endogenous transcription activator like effectors (TALEs). TALEs have effector binding elements (EBEs) in the promoter region of the OsSWEET genes. Out of six well-known TALEs found to have EBEs in Clade III SWEET genes, four are present in OsSWEET14 gene’s promoter region. Thus, targeting the promoter of OsSWEET14 is very important for creating broad-spectrum resistance. To engineer resistance against bacterial blight, we established CRISPR-Cas9 mediated genome editing in Super Basmati rice by targeting 4 EBEs present in the promoter of OsSWEET14. We were able to obtain four different Super Basmati lines (SB-E1, SB-E2, SB-E3, and SB-E4) having edited EBEs of three TALEs (AvrXa7, PthXo3, and TalF). The edited lines were then evaluated in triplicate for resistance against bacterial blight by choosing one of the locally isolated virulent Xoo strains with AvrXa7 and infecting Super Basmati. The lines with deletions in EBE of AvrXa7 showed resistance against the Xoo strain. Thus, it was confirmed that edited EBEs provide resistance against their respective TALEs present in Xoo strains. In this study up to 9% editing efficiency was obtained. Our findings showed that CRISPR-Cas9 can be harnessed to generate resistance against bacterial blight in indigenous varieties, against locally prevalent Xoo strains.

Bacterial blight (BB) and bacterial leaf streak (BLS) are important diseases in Oryza sativa caused by Xanthomonas oryzae pv. oryzae (Xoo) and Xanthomonas oryzae pv. oryzicola (Xoc), respectively. In both bacteria, transcription activator-like (TAL) effectors are major virulence determinants that act by transactivating host genes downstream of effector-binding elements (EBEs) bound in a sequence-specific manner. Resistance to Xoo is mostly related to the action of TAL effectors, either by polymorphisms that prevent the induction of susceptibility (S) genes or by executor (R) genes with EBEs embedded in their promoter, and that induce cell death and resistance. For Xoc, no resistance sources are known in rice. Here, we investigated whether the recognition of effectors by nucleotide binding and leucine-rich repeat domain immune receptors (NLRs), the most widespread resistance mechanism in plants, is also able to stop BB and BLS. In one instance, transgenic rice lines harboring the AVR1-CO39 effector gene from the rice blast fungus Magnaporthe oryzae, under the control of an inducible promoter, were challenged with transgenic Xoo and Xoc strains carrying a TAL effector designed to transactivate the inducible promoter. This induced AVR1-CO39 expression and triggered BB and BLS resistance when the corresponding Pi-CO39 resistance locus was present. In a second example, the transactivation of an auto-active NLR by Xoo-delivered designer TAL effectors resulted in BB resistance, demonstrating that NLR-triggered immune responses efficiently control Xoo. This forms the foundation for future BB and BLS disease control strategies, whereupon endogenous TAL effectors will target synthetic promoter regions of Avr or NLR executor genes.

Bacterial leaf blight (BLB) of rice is considered to be a disease of economic importance as the disease causes severe yield losses in all rice growing regions. Transcription activator like effector (TALE) molecules are produced by the pathogen, Xanthomonas oryzae pv. oryzae (Xoo) bind to the effector binding element (EBE) of the promoter of SWEET gene and activates transcription of SWEET genes, making the plant susceptible to the disease.  Innate resistance to Xoo in certain rice genotypes is due to mutations in EBE present in the upstream regulatory region of SWEET genes. CRISPR–mediated targeted modification of susceptibility gene/promoter is an effective approach to develop BLB resistance in rice. The present study was an attempt to repress TALE triggered signalling via introducing indels in the EBE of OsSWEET13 gene in a local popular rice genotype, CO51 employing CRISPR/Cas9 mediated genome editing tool with a view to imparting BLB resistance. Agrobacterium-mediated transformation using immature embryos followed by regeneration resulted in four independent transformation events. Five plants representing three events were found to have one nucleotide deletion in the target sequence. These deletion mutations in EBE could potentially interfere with the binding of the corresponding TALE to confer resistance against certain strains of BLB.  Keywords: Rice, Bacterial Leaf Blight, SWEET13 gene, Effector Binding Element, CRISPR/Cas9.

TAL Effectors Drive Transcription Bidirectionally in Plants

Bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo), is one of the major diseases that impact rice production in Asia. The bacteria use transcription activator-like effectors (TALEs) to hijack the host transcription machinery and activate key susceptibility (S) genes, specifically members of the SWEET sucrose uniporters through the recognition of effector-binding element (EBEs) in the promoter regions. However, natural variations in the EBEs that alter the binding affinity of TALEs usually prevent sufficient induction of SWEET genes, leading to resistance phenotypes. In this study, we identified candidate resistance alleles by mining a rice diversity panel for mutations in the promoter of OsSWEET13 and OsSWEET14, which are direct targets of three major TALEs PthXo2, PthXo3 and AvrXa7. We found natural variations at the EBE of both genes, which appeared to have emerged independently in at least three rice subspecies. For OsSWEET13, a 2-bp deletion at the 5th and 6th positions of the EBE, and a substitution at the 17th position appear to be sufficient to prevent activation by PthXo2. Similarly, a single nucleotide substitution at position 10 compromised the induction of OsSWEET14 by AvrXa7. These findings might increase our opportunities to reduce pathogen virulence by preventing the induction of SWEET transporters. Pyramiding variants along with other resistance genes may provide durable and broad-spectrum resistance to the disease.

SummaryAs a key virulence strategy to cause bacterial leaf blight, Xanthomonas oryzae pv. oryzae (Xoo) injects into the plant cell DNA‐binding proteins called transcription activator‐like effectors (TALEs) that bind to effector‐binding elements (EBEs) in a sequence‐specific manner, resulting in host gene induction. TALEs AvrXa7, PthXo3, TalC and Tal5, found in geographically distant Xoo strains, all target OsSWEET14, thus considered as a pivotal TALE target acting as major susceptibility factor during rice–Xoo interactions. Here, we report the generation of an allele library of the OsSWEET14 promoter through stable expression of TALE‐nuclease (TALEN) constructs in rice. The susceptibility level of lines carrying mutations in AvrXa7, Tal5 or TalC EBEs was assessed. Plants edited in AvrXa7 or Tal5 EBEs were resistant to bacterial strains relying on the corresponding TALE. Surprisingly, although indels within TalC EBE prevented OsSWEET14 induction in response to BAI3 wild‐type bacteria relying on TalC, loss of TalC responsiveness failed to confer resistance to this strain. The TalC EBE mutant line was, however, resistant to a strain expressing an artificial SWEET14‐inducing TALE whose EBE was also edited in this line. This work offers the first set of alleles edited in TalC EBE and uncovers a distinct, broader range of activities for TalC compared to AvrXa7 or Tal5. We propose the existence of additional targets for TalC beyond SWEET14, suggesting that TALE‐mediated plant susceptibility may result from induction of several, genetically redundant, host susceptibility genes by a single effector.

Most Xanthomonas species translocate Transcription Activator-Like (TAL) effectors into plant cells where they function like plant transcription factors via a programmable DNA-binding domain. Characterized strains of rice pathogenic X. oryzae pv. oryzae harbor 9–16 different tal effector genes, but the function of only a few of them has been decoded. Using sequencing of entire genomes, we first performed comparative analyses of the complete repertoires of TAL effectors, herein referred to as TALomes, in three Xoo strains forming an African genetic lineage different from Asian Xoo. A phylogenetic analysis of the three TALomes combined with in silico predictions of TAL effector targets showed that African Xoo TALomes are highly conserved, genetically distant from Asian ones, and closely related to TAL effectors from the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Nine clusters of TAL effectors could be identified among the three TALomes, including three showing higher levels of variation in their repeat variable diresidues (RVDs). Detailed analyses of these groups revealed recombination events as a possible source of variation among TAL effector genes. Next, to address contribution to virulence, nine TAL effector genes from the Malian Xoo strain MAI1 and four allelic variants from the Burkinabe Xoo strain BAI3, thus representing most of the TAL effector diversity in African Xoo strains, were expressed in the TAL effector-deficient X. oryzae strain X11-5A for gain-of-function assays. Inoculation of the susceptible rice variety Azucena lead to the discovery of three TAL effectors promoting virulence, including two TAL effectors previously reported to target the susceptibility (S) gene OsSWEET14 and a novel major virulence contributor, TalB. RNA profiling experiments in rice and in silico prediction of EBEs were carried out to identify candidate targets of TalB, revealing OsTFX1, a bZIP transcription factor previously identified as a bacterial blight S gene, and OsERF#123, which encodes a subgroup IXc AP2/ERF transcription factor. Use of designer TAL effectors demonstrated that induction of either gene resulted in greater susceptibility to strain X11-5A. The induction of OsERF#123 by BAI3Δ1, a talB knockout derivative of BAI3, carrying these designer TAL effectors increased virulence of BAI3Δ1, validating OsERF#123 as a new, bacterial blight S gene.

Bacterial blight (BB) of rice caused by Xanthomonas oryzae pv. oryzae (Xoo), is an important disease in rice-growing countries, including Pakistan, where it was first reported in the mid-1970s. Transcription activator-like effectors (TALEs) play vital roles in many plant diseases caused by Xanthomonas spp.; however, Pakistani Xoo TALome diversity and their contribution to pathogenicity is largely unknown. In this study, 101 Xoo strains were screened using specific PCR primers. The genomic DNA from these strains underwent BamHI digestion and hybridized with the internal SphI fragment of PthXo1. Southern blot analysis revealed 16 to 20 putative tale fragments among the tested strains. These strains were further classified into 11 genotypes based on the number and size of the hybridizing bands. Genotypes 1, 2, 3, and 4 represented 24, 2, 51, and 17 strains, respectively. Pathogenicity assays on near-isogenic lines (NILs) containing different resistance (R) genes exhibited that CBB23 was incompatible with all tested Pakistani-Xoo genotypes, whereas IRBB5 and IRBB4 showed resistance against specific genotypes. In contrast, paddy trails on NILs containing single, double, and triple mutants of OsSWEET11a, OsSWEET13, and OsSWEET14 in the effector binding elements (EBEs) of cv. Kitaake revealed that KP-22 and LD-5 harbor novel virulent TAL effector/s. Interestingly, the expression analysis of six clade-III OsSWEET genes suggests that novel TALE/s targeting unidentified susceptibility gene/s. Altogether, this study highlights gene-for-gene relationships between tested rice lines and Pakistani-Xoo strains. This is the first report providing the diversity of TALEs and their relationship to R and S (susceptibility) genes. Further identification of novel virulent TALE/s and their cognate target/s is warranted to precisely elucidate their role in BB.

Bac thom 7 rice (BT7 rice) is one of the major elite rice cultivars in Vietnam with superior productivity and quality but very susceptible to bacterial leaf blight caused by Xanthomonas oryzae pv. oryzae. The gene OsSWEET14, belonging to the OsSWEET family which encodes sugar transport proteins, is considered to be a susceptible gene involved in the virulence of Xoo. At least three cis-elements (EBE - Effector-binding element), including Tal5, PthXo3 and AvrXa7 on BT7 OsSWEET14 promoter, bind specifically to well-known transcription activator–like effectors (TALEs) of many Asian Xoo bacterium strains. In this study, a T-DNA construct which expressed the protein-RNA complex CRISPR/Cas for editing three EBEs position on the OsSWEET14 promoter was designed. The recombinant binary vector was tested by PCR, restriction enzyme and finally sequencing and then successfully transferred to Bac thom 7 rice through Agrobacterium tumefaciens. 28 of 30 hygromycin-resistant regenerated rice lines that grew and developed normally under nethouse conditions were selected by PCR with specific primers. Among these, twelve transgenic rice lines were identified carrying one copy of the T-DNA construct. The presence of CRISPR/Cas9-induced mutations of the targeted promoter in the transgenic plants were confirmed by T7EI assay. These results provide the basis to determine the role of OsSWEET14 in the susceptibility of Bac thom 7 rice to Xanthomonas oryzae pv. oryzae -caused disease, towards the further goal of generating an improved Bac thom 7 rice variety with broad-spectrum bacterial leaf blight resistance using CRISPR/Cas9 technology.

Blight-resistant rice lines are the most effective solution for bacterial blight, caused by Xanthomonas oryzae pv. oryzae (Xoo). Key resistance mechanisms involve SWEET genes as susceptibility factors. Bacterial transcription activator-like (TAL) effectors bind to effector-binding elements (EBEs) in SWEET gene promoters and induce SWEET genes. EBE variants that cannot be recognized by TAL effectors abrogate induction, causing resistance. Here we describe a diagnostic kit to enable analysis of bacterial blight in the field and identification of suitable resistant lines. Specifically, we include a SWEET promoter database, RT–PCR primers for detecting SWEET induction, engineered reporter rice lines to visualize SWEET protein accumulation and knock-out rice lines to identify virulence mechanisms in bacterial isolates. We also developed CRISPR–Cas9 genome-edited Kitaake rice to evaluate the efficacy of EBE mutations in resistance, software to predict the optimal resistance gene set for a specific geographic region, and two resistant ‘mega’ rice lines that will empower farmers to plant lines that are most likely to resist rice blight.

Xanthomonas oryzae pv. oryzae (Xoo) strains that cause bacterial leaf blight (BLB) limit rice (Oryza sativa) production and require breeding more resistant varieties. Transcription activator-like effectors (TALEs) activate transcription to promote leaf colonization by binding to specific plant host DNA sequences termed effector binding elements (EBEs). Xoo major TALEs universally target susceptibility genes of the SWEET transporter family. TALE-unresponsive alleles of clade III OsSWEET susceptibility gene promoter created with genome editing confer broad resistance on Asian Xoo strains. African Xoo strains rely primarily on the major TALE TalC, which targets OsSWEET14. Although the virulence of a talC mutant strain is severely impaired, abrogating OsSWEET14 induction with genome editing does not confer equivalent resistance on African Xoo. To address this contradiction, we postulated the existence of a TalC target susceptibility gene redundant with OsSWEET14. Bioinformatics analysis identified a rice locus named ATAC composed of the INCREASED LEAF INCLINATION 2 (ILI2) gene and a putative lncRNA that are shown to be bidirectionally upregulated in a TalC-dependent fashion. Gain-of-function approaches with designer TALEs inducing ATAC sequences did not complement the virulence of a Xoo strain defective for SWEET gene activation. While editing the TalC EBE at the ATAC loci compromised TalC-mediated induction, multiplex edited lines with mutations at the OsSWEET14 and ATAC loci remained essentially susceptible to African Xoo strains. Overall, this work indicates that ATAC is a probable TalC off-target locus but nonetheless documents the first example of divergent transcription activation by a native TALE during infection.

Bacterial blight (BB), caused by Xanthomonas oryzae pv. oryzae (Xoo), is a widespread and destructive disease in rice production. Previously, we cloned an executor R gene, Xa7, which confers durable and broad-spectrum resistance to BB. Here, we further confirmed that the transcription activator-like effector (TALE) AvrXa7 in Xoo strains could directly bind to the effector-binding element (EBE) in the promoter of the Xa7 gene. Other executor R genes (Xa7, Xa10, Xa23, and Xa27) driven by the promoter of the Xa7 gene could be activated by AvrXa7 and trigger the hypersensitive response (HR) in tobacco leaves. When the expression of the Xa23 gene was driven by the Xa7 promoter, the transgenic rice plants displayed a similar resistance spectrum as the Xa7 gene, demonstrating that the disease resistance characteristics of executor R genes are mainly determined by their induction patterns. Xa7 gene is induced locally by Xoo in the infected leaves, and its induction not only inhibited the growth of incompatible strains but also enhanced the resistance of rice plants to compatible strains, which overcame the shortcomings of its race-specific resistance. Transcriptome analysis of the Xa7 gene constitutive expression in rice plants displayed that Xa7-mediated disease resistance was related to the biosynthesis of lignin and thus enhanced resistance to Xoo. Overall, our results provided novel insights and important resources for further clarifying the molecular mechanisms of the executor R genes.

Xanthomonas transcription activator-like (TAL) effectors promote disease in plants by binding to and activating host susceptibility genes. Plants counter with TAL effector-activated executor resistance genes, which cause host cell death and block disease progression. We asked whether the functional specificity of an executor gene could be broadened by adding different TAL effector binding elements (EBEs) to it. We added six EBEs to the rice Xa27 gene, which confers resistance to strains of the bacterial blight pathogen Xanthomonas oryzae pv. oryzae (Xoo) that deliver the TAL effector AvrXa27. The EBEs correspond to three other effectors from Xoo strain PXO99(A) and three from strain BLS256 of the bacterial leaf streak pathogen Xanthomonas oryzae pv. oryzicola (Xoc). Stable integration into rice produced healthy lines exhibiting gene activation by each TAL effector, and resistance to PXO99(A) , a PXO99(A) derivative lacking AvrXa27, and BLS256, as well as two other Xoo and 10 Xoc strains virulent toward wildtype Xa27 plants. Transcripts initiated primarily at a common site. Sequences in the EBEs were found to occur nonrandomly in rice promoters, suggesting an overlap with endogenous regulatory sequences. Thus, executor gene specificity can be broadened by adding EBEs, but caution is warranted because of the possible coincident introduction of endogenous regulatory elements.

Xa7, a new executor R gene that confers durable and broad-spectrum resistance to bacterial blight disease in rice